Aptamers and methods for their in vitro selection and uses thereof

ABSTRACT

The present method is an improved in vitro selection protocol that relies on magnetic separations for DNA aptamer production that is relatively easy and scalable without the need for expensive robotics. The ability of aptamers selected by this method to recognize and bind their target protein with high affinity and specificity, and detail their uses in a number of assays is also described. Specific TTF1 and His6 aptamers were selected using the method described, and shown to be useful for enzyme-linked assays, Western blots, and affinity purification.

CROSS REFERENCE TO RELATED APPLICATION

This application relates to and claims benefit of Provisional PatentApplication Ser. No. 60/500,800, filed Sep. 4, 2003, and ProvisionalPatent Application Ser. No. 60/584,591, filed Jun. 30, 2004, and is adivisional application to U.S. patent application Ser. No. 10/934,856,filed on Sep. 3, 2004, to be issued as U.S. Pat. No. 7,329,742, all ofwhich are hereby incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM APPENDIX

Applicants assert that the attached paper copy of the Sequence Listingfor the utility application, “Aptamers and methods for their in vitroselection and uses thereof,” claiming priority to U.S. ProvisionalPatent Application No. 60/500,800 filed Sep. 4, 2003, and ProvisionalPatent Application Ser. No. 60/584,591, filed Jun. 30, 2004, isidentical to the Sequence Listing in computer readable form alsoattached as a .txt file, “IB1929_SEQLISTING8.txt”, created on Jul. 6,2007, as required by 37 CFR 1.821(c), both of which are herebyincorporated by reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made during work supported by the U.S. Department ofEnergy at Lawrence Livermore National Laboratory under contract No.W-7405-ENG-48, Lawrence Berkeley National Laboratory under contract No.DE-AC03-76SF00098, and Los Alamos National Laboratory under contract No.W-7405-ENG-36. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to aptamers and methods for the selection andgeneration of aptamers for use in such fields of application asproteomics, protein detection and purification, drug design anddevelopment, and protein purification and capture reagents.

2. Prior Relevant Art

The future success of proteomics depends on its ability to follow in thefootsteps of genomics, where the development of new technologiesgenerated an abundance of sequence data enabling researchers to probeproblems that relate to the entire nucleic acid component of the cell.For the promise of proteomics to be realized, new tools are needed thatwill enable large-scale investigations of protein structure, function,and interactions. Significant progress has been made in proteomictechnology development in many areas, including high-throughput genecloning, protein production, mass spectrometry, 2-D PAGE, andmicrofluidics to allow large-scale proteomics to proceed.

One important set of tools that has been improved with moderate successare affinity reagents that function as antibodies to serve as proteinprobes. Affinity molecules that specifically bind proteins of interestcan detect bound proteins in a protein microarray, or capture proteincomplexes for functional identification. Often these molecules can alterbiological activity due to their binding and inhibit criticalinteractions by sterically blocking access to active sites andinteraction surfaces, and thus present an opportunity to serve asfunctional probes as well as therapeutics. Traditionally, antibodieshave satisfied the demand for such ligands, however as recombinantprotein production gains throughput and pharmaceutical targetrepertoires expand, the ability to efficiently generate antibodiesquickly falls short.

Several alternatives to antibodies have been investigated, such assingle chain antibodies (scFv), peptides displayed on protein domainscaffolded surfaces, peptides, and peptoids (synthetic peptides). Eachof these alternatives has drawbacks that limit their uses, such asproblems of stability in varying conditions (ionic strength,temperature, and pH) and of low affinity, making some antibodyalternatives ineffective for detecting proteins under many conditions.

The use of aptamers as protein affinity reagents offers advantages overthe use of antibodies. Nucleic acids are easily synthesized or amplifiedby PCR; therefore a vast supply of consistent quality is available.Also, nucleic acids can easily be modified to incorporate tags, such asbiotin or fluorescent molecules, for detection and/or immobilization.Additionally, aptamers are smaller (<25 kDa) and more stable thanantibodies. Moreover, unlike the requirement of milligram quantities ofprotein or peptide for antibody production, only microgram quantities ofprotein or peptide are required for aptamer selection. These properties,coupled to the present technology available for DNA microarrays, makeaptamers very suitable for use in protein microarrays as a ligand, orfor detecting proteins bound to a chip surface (See Walter G, Bussow K,Lueking A, Glokler J. (2002) High-throughput protein arrays: prospectsfor molecular diagnostics. Trends Mol. Med. June; 8(6):250-3).

The idea of using single stranded nucleic acids (aptamers) as affinitymolecules for proteins has shown modest progress. See Tuerk C, Gold L.(1990) Systematic evolution of ligands by exponential enrichment: RNAligands to bacteriophage T4 DNA polymerase. Science. August 3;249(4968):505-10; Ellington A D, Szostak J W. (1990) In vitro selectionof RNA molecules that bind specific ligands. Nature. August 30;346(6287):818-22; and Ellington A D, Szostak J W. (1992) Selection invitro of single-stranded DNA molecules that fold into specificligand-binding structures. Nature. February 27; 355(6363):850-2. Theconcept is based on the ability of short oligomer (20-80 mer) sequencesto fold, in the presence of a target, into unique 3-dimensionalstructures that bind the target with high affinity and specificity.Aptamers are generated by a process that combines combinatorialchemistry with in vitro evolution, commonly known as SELEX (SystematicEvolution of Ligands by Exponential Enrichment). Following theincubation of a protein with a library of DNA or RNA sequences(typically 10¹⁴ molecules in complexity) protein-DNA complexes areisolated, the DNA is amplified, and the process is repeated until thesample is enriched with sequences that display high affinity for theprotein of interest. Since the selection pressure is high affinity forthe target, aptamers with low nanomolar affinities may be obtained.Aptamers offer advantages over protein-based affinity reagents becausenucleic acids possess increased stability, ease of regeneration (PCR oroligonucleotide synthesis), and simple modification for detection andimmobilization.

Although SELEX appears to be technically very simple, small alterationsto a protocol can have a large impact on the success of generatingaptamers. Perhaps this explains why thirteen years since its firstcitation in the literature, only approximately forty protein-detectingaptamer sequences have been published, and very few have been wellcharacterized. Although high-throughput methods for aptamer productionhave been published, most require expensive robotics and have notproduced aptamers against large numbers of diverse targets (Cox J C,Rajendran M, Riedel T, Davidson E A, Sooter L J, Bayer T S,Schmitz-Brown M, Ellington A D. (2002) Automated acquisition of aptamersequences. Comb Chem High Throughput Screen. June; 5(4):289-99).

Many variations in aptamer production protocols have been described inwhich the method of protein target partitioning seems to vary the most.Unbound DNA molecules have been removed from target proteins via: 1)filtration on a membrane (Ellington A D, Szostak J W. (1992) Selectionin vitro of single-stranded DNA molecules that fold into specificligand-binding structures. Nature. February 27; 355(6363):850-2); 2)column chromatography, in which the targets are bound to a matrix, suchas sepharose, using a covalent linkage or an affinity tag (Ylera F, LurzR, Erdmann V A, Furste J P. (2002) Selection of RNA aptamers to theAlzheimer's disease amyloid peptide. Biochem Biophys Res Commun.February 8; 290(5):1583-); and 3) binding of the protein to the wells ofa microtiter plate (Drolet D W, Jenison R D, Smith D E, Pratt D, Hicke BJ. (1999) A high throughput platform for systematic evolution of ligandsby exponential enrichment (SELEX). Comb Chem High Throughput Screen.October; 2(5):271-8).

Gorenstein, et al, in U.S. Pat. No. 6,423,493, describe a randomcombinatorial selection method for the construction of oligonucleotideaptamers in which nuclease resistance is conferred by the inclusion ofmodified nucleotides. The modified nucleotides are incorporated duringPCR amplification to form achiral modified oligonucleotides.Thio-substituted aptamers are provided that bind tightly to the nuclearfactor for human IL6 (NF-IL6).

Kwagh, et al., in U.S. Pat. No. 6,515,120, describe a method forsequencing and structurally characterizing a polymeric biomolecule usingaptamers and also describes aptamers that recognize and bind to AMP,dAMP, GMP, dGMP, CMP and dCMP.

In U.S. Pat. No. 6,180,348, Li describes a method that makes use ofmagnetic separation to identify an aptamer which specifically binds to atarget molecule of interest. A method for identifying oligomersequences, optionally comprising modified bases, which specifically bindtarget molecules such as serum proteins, kinins, eicosanoids andextracellular proteins is described by Griffin, et al in U.S. Pat. No.5,756,291. The method is used to generate aptamers that bind to serumFactor X, PDGF, FGF, ICAM, VCAM, E-selectin, thrombin, bradykinin, PGF2and cell surface molecules.

SUMMARY OF THE INVENTION

The invention provides for a method for obtaining an aptamer having highaffinity to a target molecule, comprising: (a) preparing a targetmolecule with a polyhistidine affinity tag for magnetic beads; (b)binding the target molecule to magnetic beads and contacting the targetmolecule with a library of aptamer sequences to allow binding of aptamersequences to the target molecule thus forming bead-target-aptamersequence complexes, wherein the aptamer sequences are comprised ofdegenerate sequences; (c) separating bead-target-aptamer sequencecomplexes from non-binding aptamer sequences by retaining the targetmolecule on its bead and removing unbound aptamer sequences; then (d)separating target-bound aptamer sequences from said magnetic beads toform a pool of binding aptamer sequences; (e) amplifying the bindingaptamer sequences; and (f) iteratively repeating steps (b) through (d) asufficient number of times to result in identification of at least oneaptamer sequence having high affinity for the target molecule.

The method further comprising: subcloning the high affinity aptamersequence into a plasmid and transforming E. coli; isolating said plasmidcontaining said aptamer sequence from transformed E. coli; andamplifying and sequencing said cloned aptamer sequence.

In one embodiment, the magnetic beads are Ni-coated. In anotherembodiment, the targets are hexahistidine-tagged proteins.

In one aspect, an aptamer of the present invention is a single strandedDNA sequence, although RNA or other amplifiable nucleic acid basedpolymers can be used. In a preferred embodiment, the aptamer sequence isdegenerate random sequence about 20-50 bases long. In another embodimentthe aptamer further comprises the degenerate sequence flanked by fixedsequences. In a preferred embodiment, the flanking fixed sequencespermit ligation-independent cloning. In another embodiment, the flankingfixed sequences are primer sequences that can be biotinylated

In another aspect, a library of 10⁸ to 10¹⁵ random aptamers is firstgenerated and incubated with the target molecule. The unbound aptamersare washed away. The aptamers bound to the targets are eluted andamplified by PCR to form a new pool for another round of binding to thetargets.

In another embodiment, the method can further comprise: subcloning thehigh affinity aptamer sequence into a plasmid and transforming E. coliwith the affinity aptamer sequence; isolating the plasmid clonecontaining the aptamer sequence from the transformed E. coli; andamplifying and sequencing the cloned aptamer sequence. In a preferredembodiment, the plasmid is selected from the group consisting ofpUC18LIC and pET30XaLIC, and amplification is by rolling circleamplification.

In another embodiment, the magnetic beads are streptavidin coated andthe targets are His6 peptides or proteins tagged with biotin to generateaptamers against a His6 peptide. In this embodiment, the His6 peptidetagged with biotin facilitates the attachment of the His6 peptide tostreptavidin magnetic beads.

The invention also describes novel nucleic acid molecules or aptamersgenerated by the methods of the invention. The invention furtherprovides for aptamers generated by the method described herein, whereinthe aptamers have high binding affinity to thyroid transcription factor1 (TTF1) protein. The TTF1 aptamer comprising a sequence having at least90% homology to a sequence selected from the group consisting of SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 13.The aptamer sequence can further comprise flanking fixed sequences. Inone embodiment, the flanking fixed sequences are primers such as SEQ IDNO: 17 and SEQ ID NO: 18, wherein one of the primers is biotinylated.

In another embodiment, aptamers were generated to a polyhistidine(His6). These His6 aptamers, the sequences of which are hereindescribed, are useful for the capture and detection of proteins thatcontain at least 5, normally 6 or more histidine residues (His6) fusedto target peptides, proteins and biomolecules. The His6 aptamerscomprising a sequence having at least 90% homology to a sequenceselected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, andSEQ ID NO: 16. The aptamer sequence can further comprise flanking fixedsequences. In one embodiment, the flanking fixed sequences are primerssuch as SEQ ID NO: 17 and SEQ ID NO: 18, wherein one of the primers isbiotinylated.

The invention further contemplates that these aptamer sequences may bevaried in the degenerate sequence region, by up to 20 percent, morepreferably by 10 percent, most preferably by 5 percent, while retainingthe functional properties as described herein.

The invention also provides a method for obtaining a His6 aptamer havinghigh affinity to a target molecule, comprising: (a) preparing a targetpolyhistidine molecule with an affinity tag for magnetic beads; (b)binding the target molecule to magnetic beads and contacting the targetmolecule with a library of aptamers of different sequences to allowbinding of aptamer sequences to the target molecule thus formingbead-target-aptamer sequence complexes; (c) separatingbead-target-aptamer sequence complexes from non-binding aptamers byretaining the target molecule on its bead and removing unbound aptamers;then (d) separating target-bound aptamers from said magnetic beads toform a pool of binding aptamers; (e) amplifying the binding aptamers;and (f) iteratively repeating steps (b) through (d) a sufficient numberof times to result in identification of at least one aptamer sequencehaving high affinity for the target molecule. In one embodiment, themagnetic beads are streptavidin-coated and the target polyhistidinemolecule is biotinylated. The invention further provides for an aptamerisolated by the method described, wherein the aptamer effectively bindsto a polyhistidine residue tag. In another aspect, the sequence of theaptamer is at least 80% homologous to SEQ ID NO: 14, 15 or 16.

One embodiment of the present invention involves preparing a library ofprotein-binding aptamers for use with complex mixtures of proteins.Because of the stability of the present aptamers, they may beimmobilized on chips or microarrays. They may be used to isolate andpurify proteins as well.

The invention further provides for a method of protein purificationcomprising the steps of: (a) providing an affinity column, wherein theaffinity tag is an aptamer isolated by the methods described; and (b)applying a crude extract or culture from which a target protein is to beisolated. In one embodiment, the affinity tag is a His6 aptamer and thetarget protein has a polyhistidine tag. In a preferred embodiment, theHis6 aptamer is at least 80% homologous to SEQ ID NO: 14, 15 or 16.

It is further an object of the disclosed invention to provide aptamersthat bind sequential histidine amino acid residues or to sequentialhistidine amino acid residues fused to any other amino acid residues orprotein. The invention also contemplates the use of these His6 aptamersin biological applications, including the use with solid supports foraffinity resins, magnetic or polymer beads, as a diagnostic detectionreagent, to capture or immobilize reagents for diagnostic, detection orquantitative studies.

The invention provides for an apparatus comprising: (a) a solid support;and (b) an His6 aptamer or an array of His6 aptamers attached to thesolid support. In one aspect, the solid support is comprised of glass,metal silicon, ceramic or polymer. The apparatus further comprises apeptide or protein bound to the aptamer. The peptide or protein binds tothe His6 aptamer by means of a polyhistidine tag. In a preferredembodiment, the His6 aptamer is at least 80% homologous to SEQ ID NO:14, 15 or 16.

In another aspect, the solid support is coated with at least onematerial selected from the group consisting of gold, avidin,streptavidin, carboxymethyl groups, dextran or collagen. In oneembodiment, the solid support is coated with streptavidin and theaptamer is biotinylated, whereby the aptamer attaches to the solidsupport through the binding of biotin and the streptavidin-coating.

In one embodiment, the His6 aptamer is attached to the solid support bymeans of an oligonucleotide. In this embodiment, the oligonucleotide isbiotinylated and attaches to the solid support through the binding ofbiotin and the streptavidin-coating. In one aspect, the aptamer wouldfurther comprise flanking primer sequences, whereby at least 3 bases ofthe flanking primer sequences of the aptamer has base complementarity tothe oligonucleotide. In another aspect, the invention further comprisinga peptide or protein bound to the aptamer by means of a polyhistidinetag.

It is also an object of the invention to provide a method of capturing amolecule of interest, comprising the steps of: (a) providing a solidsupport having an aptamer or array of aptamers attached to the support,wherein the aptamer comprises a sequence having at least 90% homology toSEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; (b) applying a samplecontaining a molecule of interest having a polyhistidine tag to thesupport; (c) capturing the molecule of interest.

In one aspect, the solid support comprises glass, metal ceramic orpolymeric materials. The solid support can be selected from the groupconsisting of steel, gold, silver, aluminum, copper, silicon, glass,polyethylene, polypropylene, polyamide, and polyvinylidenefluoride, andcombinations thereof. In another aspect, the solid support is coatedwith a material to facilitate the attachment, binding, hybridization,immobilization or interaction of the aptamer on the surface. In apreferred embodiment, the coating comprises gold, carboxymethylation,dextran, collagen, avidin or streptavidin.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the method of constructing an N-terminal 6×His tag vectorfor Ligation Independent Cloning (LIC).

FIG. 2 shows the High-throughput Cloning Method with LIC.

FIG. 3 is a cartoon which shows the In Vitro Selection Protocol for theproduction of ssDNA ligands or aptamers that bind targets with highaffinity and specificity.

FIG. 4 shows the selected aptamers to TTF1 enriched from 15 rounds ofselection. The 40 base variable region of each aptamer sequence is shown(5′-3′). The number of times that each sequence was obtained from atotal of 30 isolates is displayed on the right. A consensus sequencefound in two of the aptamers is underlined.

FIG. 5 is a graph showing the determination of specificity of aptamersusing an enzyme-linked assay. Combinations of TTF1, HOX4, and BSAprotein and either a TTF1 aptamer “A” or HOX4 aptamer (indicated by plussigns below the graph) were evaluated for their binding activity andcross-reactivity. The data are from triplicate samples.

FIG. 6 comprises four graphs showing the determination of the affinitiesof aptamers for TTF1 using surface plasmon resonance. (A) Sensorgrams ofthe binding response to aptamer “A” measured for concentrations of 2.5,5, 10, 20, 40, 100 nM TTF1 analyte. The K_(D)=3.36×10−9 M as determinedfrom a global fit of the kinetic simultaneous k_(a)/k_(d) model,assuming Langmuir (1:1) binding, and Chi²=14.1. (B) Plot of thesteady-state affinity for “A” using the Req values derived fromsensorgrams in (A) fitted locally. The K_(D)=5.14×10−9 M as determinedfrom the steady state affinity model. (C) Sensorgrams of the bindingresponses to aptamer “C” measured for concentrations of 3, 12, 31, 62.5,125, 250 nM TTF1 analyte. The K_(D)=3.25×10−8 M as determined from aglobal fit of the kinetic simultaneous k_(a)/k_(d) model, assumingLangmuir (1:1) binding, and Chi²=10.9. (D) Plot of the steady-stateaffinity for “C” using the Req values derived from sensorgrams in (C)fitted locally. The K_(D)=6.56×10−8 M as determined from the steadystate affinity model.

FIG. 7 is a series of three photographs (A-C) of a comparison of thespecificity of TTF1 aptamer “A” to a monoclonal anti-Penta-His antibodyusing protein blot analysis. Lane 1 contains cleared lysate from E. coliexpressing the HOX4 homeodomain. Lane 2 contains purified HOX4homeodomain protein (marked with an arrow). Lane 3 contains clearedlysate from E. coli expressing TTF1. Lane 4 contains purified TTF1protein (marked with an arrow). (A) 4-20% SDS-PAGE stained with GelCodeblue. (B) Blot of material shown in (A) probed with an anti-PentaHismonoclonal antibody. (C) Blot of material shown in (A) probed with thebiotinylated TTF1 aptamer “A”. Note that the lower dark bands in lanes 1and 3 of (C) were detected by the secondary probe, Streptavidin-HRP,(not shown).

FIG. 8 is a photograph of a nine lane SDS-PAGE analysis of aptameraffinity purification of TTF1 protein from E. coli lysates usingbiotinylated TTF1 aptamer “A” immobilized on streptavidin magneticbeads. Lane 1 contains cleared lysate from E. coli expressing theprotein of interest, and lane 2 contains the cleared lysate spiked withNi-NTA-purified TTF1 protein (lane 3). Material in lane 4 was from 10min binding and 2 h elution at 4° C. Material in lane 6 was from 30 minbinding and 15 min elution at room temperature. Material in lane 8 wasfrom 5 min binding and 5 min elution at room temperature. After eachelution with Benzonase, any remaining protein was removed from theaptamer with 0.1% SDS (Lanes 5, 7 and 9).

FIG. 9 is a copy of the display output showing the aptamer sequencealignment for the His6 aptamers 6H1, 6H5 and 6H7.

FIG. 10 is a schematic showing various capture methods for SPRapplications including (A) traditional methods using an antibody thatbinds to a His6 tag, (B) use of a biotinylated aptamer on a streptavidinchip that binds to a His6 tag, and (C) use of a biotinylatedlinker/primer on a streptavidin (SA) chip that binds to a fixed portionof the aptamer by base complementarity.

FIG. 11 is a graph showing the association and dissociation of 1293His-tagged protein from SA-6H5 aptamer vs. 6His antibody.

FIG. 12 is a graph showing dissociation curves for 10, 100, and 1000 nM1293 His-tagged protein from 6His antibody chip.

FIG. 13 is a graph showing dissociation curves for 100 nM 1293His-tagged protein from 6His antibody chip and 1000 nM 1293 His-taggedprotein from SA-6H5 aptamer chip. These curves show similar dissociationrates despite the 10-fold higher concentration of protein from theaptamer sample.

FIG. 14 is a graph showing the baseline drift of 6H5 aptamer immobilizedon SA chip via biotin vs. 6H5 aptamer immobilized via complementarylinker/primer.

FIG. 15 is a sensorgram of TTF-1 aptamer binding and regeneration fromlinker/primer and TTF-1 protein binding and regeneration fromlinker/aptamer complex.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Introduction

The present invention provides for an in vitro ssDNA selection methodthat utilizes tagged target molecules bound to magnetic beads to screenfor high affinity binding aptamers from a library of degeneratesequences. The invention also describes novel nucleic acid molecules oraptamers generated by the methods of the invention. The invention alsocontemplates the use of these His6 aptamers in biological applications,including the use with solid supports for affinity resins, magnetic orpolymer beads, as a diagnostic detection reagent, to capture orimmobilize reagents for diagnostic, detection or quantitative studies

As used herein, the terms “aptamer(s)” or “aptamer sequence(s)” aremeant to refer to single stranded nucleic acids (RNA or DNA) whosedistinct nucleotide sequence determines the folding of the molecule intoa unique three dimensional structure. Aptamers comprising 15 to 120nucleotides can be selected in vitro from a randomized pool ofoligonucleotides (10¹⁴-10¹⁵ molecules). The “aptamers or aptamersequences” comprise a degenerate sequence, and can further comprisefixed sequences flanking the degenerate sequence. The term “aptamer” asused herein further contemplates the use of both native and modified DNAand RNA bases, e.g. beta-D-Glucosyl-Hydroxymethyluracil.

As used herein, a polynucleotide or fragment thereof is “substantiallyhomologous” (or “substantially similar”) to another if, when optimallyaligned (with appropriate nucleotide insertions or deletions) with theother polynucleotide (or its complementary strand), using BLASTN(Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J.(1990) “Basic local alignment search tool.” J. Mol. Biol. 215:403-410)there is nucleotide sequence identity in at least about 80%, preferablyat least about 90%, and more preferably at least about 95-98% of thenucleotide bases. To determine homology between two differentpolynucleotides, the percent homology is to be determined using theBLASTN program “BLAST 2 sequences”. This program is available for publicuse from the National Center for Biotechnology Information (NCBI) overthe Internet (Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2sequences—a new tool for comparing protein and nucleotide sequences”,FEMS Microbiol Lett. 174:247-250). The parameters to be used arewhatever combination of the following yields the highest calculatedpercent homology (as calculated below) with the default parameters shownin parentheses:

Program—blastn

Reward for a match—0 or 1 (1)

Penalty for a mismatch—0, −1, −2 or −3 (−2)

Open gap penalty—0, 1, 2, 3, 4 or 5 (5)

Extension gap penalty—0 or 1 (1)

Gap x_dropoff—0 or 50 (50)

Expect—10

Word size—11

Filter—low complexity.

II. In Vitro Selection of Aptamer Sequences

The present aptamer selection method generally comprises the followingsteps: (1) interacting a target molecule (e.g. recombinant protein,peptide, carbohydrate, lipid, glycolipid, etc.) comprising a tag forbinding to magnetic beads with a library of aptamer sequences to allowspecific interaction and binding of aptamer sequences to the target thusforming bead-target-aptamer sequence complexes; (2) separatingbead-target-aptamer sequence complexes from non-binding aptamersequences using the magnetic beads leaving only the bead-target-aptamersequence complexes; (3) releasing target-aptamer sequence from saidmagnetic beads; (4) amplifying aptamer sequences that bound to thetarget; and (5) repeating steps (1) through (4) a sufficient number oftimes to result in an aptamer sequence having high affinity for thetarget.

The method can further comprise the following steps: (1) subcloning thehigh affinity aptamer sequence into a plasmid and transforming E. coli;(2) isolating said plasmid containing said aptamer sequence fromtransformed E. coli; (3) amplifying and sequencing said cloned aptamersequence.

In a specific preferred embodiment, the method was optimized to useNi-coated magnetic beads to provide a universal support forhexahistidine-tagged protein targets as well as to facilitate the rapidpartitioning of protein-aptamer complexes from unselected sequencepools. During the course of optimizing the method an enrichment ofsequences that were not unique to a particular protein was observed, andtherefore counter-selection steps against Ni-coated magnetic beads toprevent enrichment of aptamers that recognize the beads only wereincorporated. The number of PCR cycles was also optimized to avoidoveramplification and mis-annealed products. The stringency of theselection can be controlled by adjusting the target proteinconcentrations, the incubation times, the concentrations of solutionsand the washes.

The preferred method was also used to facilitate DNA aptamer productionand characterization of aptamers that recognize thyroid transcriptionfactor 1 (TTF1), a member of the NK homeodomain transcription factors,and aptamers to the polyhistidine tag to recognize His6 tagged proteinsand peptides.

A. Target Molecules

In the present DNA aptamer selection method, a target molecule can beany molecule capable of forming a complex with an oligonucleotide,including, but not limited to, peptides, proteins, enzymes, receptors,antibodies, hormones, glycoproteins, polymers, polysaccharides, nucleicacids, carbohydrates, lipids, sphingolipids, small organic compoundssuch as drugs, dyes, metabolites, cofactors, transition state analogsand toxins.

Specific target molecules of interest include molecules of biologicaland physiological relevance in both prokaryotic and eukaryoticorganisms, particularly mammals. Examples of such biologicallysignificant molecules in mammals include, but are not limited to,erythropoietin, tissue plasminogen activator, granular colonystimulating factor (G-CSF), growth hormone (GH), endostatin (O'Reilly etal., (1997) Cell 88:277-285), interferons, interleukins, chemokines (Shiet al., (1997) FASEB J. 11: 1330; Bubrovsky et al., (1996) PNAS, USA92:700-709), enzymes such as SOD (Yoshikai et al., (1995) Cancer Res.55(8) 1617-1620) and amylase, antibodies (particularly the constant “Fc”regions thereof), OKT3 (Ho et al., (1998) Science 280:1866-1867), serumproteins (e.g., Factor VIII (Papadopulos-Eleopulos et al., (1990)Genetica 95:35-50), Factor VIX, plasminogen, antithrombin III (Jones etal., (1992) Br. J. Cancer 66:744-747), albumin, protein C (Griffin etal., (1993) Blood 82:1989-93), etc.), and vaccines (e.q., HbsAg (Daviset al., (1994) Vaccine 12:1503-1509), etc.). The physiologicalsignificance of most of these, and many other molecules, may similarlybe found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 8.sup.th ed., (1990) Pergamon Press, Elmsford, N.Y. Thoseof skill in the art will appreciate that a virtually unlimited number ofother target molecules may also be used with the claimed methods.

For several of the Examples, thyroid transcription factor 1 (TTF1) waschosen as the target molecule. It was recently learned that TTF1 is ahighly specific marker for primary lung adenocarcinomas, and antibodiesagainst TTF1 have been recommended to be included in a panel ofantibodies for the differential diagnosis between primary and metastaticadenocarcinomas of the lung (Reis-Filho J S, Carrilho C, Valenti C,Leitao D, Ribeiro C A, Ribeiro S G, Schmitt F C. (2000) Is TTF1 a goodimmunohistochemical marker to distinguish primary from metastatic lungadenocarcinomas? Pathol Res Pract 196(12):835-40). Therefore, the TTF1aptamers described herein may be a valuable diagnostic tool for diseasessuch as primary lung adenocarcinoma.

B. Magnetic Beads for Immobilization of Tagged Targets

Upon choosing the desired target molecule, the target molecule should betagged to allow immobilization to the magnetic beads. The use of a tagfor immobilization promotes the proper orientation of proteins uniformlyon a bead surface, and reduces the chances of selection towardcontaminants. Magnetic beads are an optimal solid support for parallelprocessing of both proteins and nucleic acids. Microliter amounts ofmagnetic beads with bound target molecules can be rapidly partitionedfrom unselected material, stringently washed, and subsequently eluted.

Magnetic beads that are coated with molecules having a specific bindingpartner that can be used as a tag, include, but are not limited to,antibodies, enzymes, antigens, sugars, saccharides, small molecules,amino and polar groups.

In one embodiment, the target molecules are tagged by a commonly usedtag such as, a polyhistidine, such as hexahistidine (His6), which bindsto nickel (Ni), or streptavidin which binds to biotin. While varioustags for binding to affinity supports are known, a preferred tag in thiscase is poly-histidine for binding to a magnetic bead. Hexahistidinetags are widely used in recombinant protein production. For example, theinventors have described an efficient protein production pipeline forhigh-throughput generation of His-tagged proteins in E. coli in Doyle SA, Murphy M B, Massi J M, and Richardson P M (2002) High-ThroughputProteomics: A Flexible and Efficient Pipeline for Protein Production. J.Proteome Research December; 1(6):531-536, which is hereby incorporatedby reference. In a preferred embodiment, Ni-NTA magnetic beads are usedfor the immobilization of His-tagged protein targets during selection.

In another embodiment, targets can be labeled with a tag such as biotinwhich permits the use of streptavidin-coated magnetic beads which arewidely available, such as Streptavidin-coated DYNAL spheres M280 (DynalBiotech, Inc., Lake Success, N.Y.). The target molecule can be easilylinked or conjugated to streptavidin-coated magnetic beads due to thestrong interaction between biotin and streptavidin.

The target-bound magnetic beads can be prepared by first equilibrating aslurry (approximately 45 μg capacity) of magnetic beads into a bufferpreferably having a salt concentration of pH 7.0 to 8.0, preferablyabout pH 7.5. A suitable solvent, for example, is PBS-T (50 mM K₂HPO₄,pH 7.5, 150 mM NaCl, 0.05% TWEEN 20). The equilibrated beads should beresuspended in the buffer then purified target is added and mixed. Thebead-bound target can then washed and diluted with buffer and stored at4° C.

C. Library of Aptamer Sequences

In one aspect of the invention, a library of 10⁸ to 10¹⁵ random aptamersis first generated and incubated with the target molecule. In apreferred embodiment, the library should contain at least 6×10¹⁴,preferably approximately 1×10¹⁵ aptamer sequences. In a preferredembodiment, these aptamer sequences are comprised of degenerateoligonucleotide sequences about 20 to 50 base pairs in length, morepreferably about 30 to 40 base pairs in length.

The aptamer sequences may further comprise fixed flanking sequences. Ina preferred embodiment, the fixed flanking sequences are primersequences flanking the degenerate oligonucleotide sequence on both ends.The primer sequences can be for use in downstream steps including, butnot limited to, hybridization, replication, amplification, ligation andsequencing. In a preferred embodiment, the length of the entire aptamersequence including the flanking fixed sequences is about 40 to 120 baseslong, more preferably between 70-90 bases long, and most preferablybetween 75-85 bases long.

The entire aptamer sequence including the fixed sequences can be madeusing commercial oligonucleotide synthesis, with the degenerate portionsof the aptamer sequences made preferably through completely randomsynthesis, but these degenerate sequences can also be made degenerate bycreating specific degenerate sequences.

Any primer sequence suitable for PCR, cloning, sequencing and otherdownstream steps can be used as a flanking sequence. In one embodiment,the flanking sequences are primers for amplification. For example,primers such as 5′-GGTATTGAGGGTCGCATC-3′ (SEQ ID NO: 17) and5′-GATGGCTCTAACTCTCCTCT-3′ (SEQ ID NO: 18), can be used. The entireaptamer sequence would be as follows:5′-GGTATTGAGGGTCGCATC-N-GATGGCTCTAACTCTCCTCT-3′ (SEQ ID NO: 1), where Nrepresents the degenerate sequence.

Primers that anneal to the 5′ and 3′ sequences flanking the degenerateregion can also be used during selection and cloning. In a preferredembodiment, the flanking sequences are primers suitable for facilitatingan improved cloning procedure in a vector that permits quick cloningsuch as ligation independent cloning (LIC). Ligation independent cloning(LIC) refers to a cloning procedure that does not require restrictionenzymes, DNA ligase, or alkaline phosphatase. The 5′ ends of the primersused to generate the clonable PCR fragments contain an additionalsequence (of about 12 nt) lacking dCTP. As a result, the amplificationproducts include 12 nt sequences lacking dGTP at their 3′ ends. The 3′terminal sequence can be removed by the action of the 3′→5′ exonucleaseactivity of T4 DNA polymerase in the presence of dGTP, leading tofragments with 5′ extending single stranded (ss) tails of a definedsequence and length. Similarly, the entire plasmid vector is amplifiedwith primers homologous to sequences in the multiple cloning site. Thevector oligos have additional 12 nt tails complementary to the tailsused for fragment amplification, permitting the creation of ss-ends withT4 DNA polymerase in the presence of dCTP. Circularization can occurbetween vector molecules and PCR fragments as mediated by the 12-nttails, but not in mixtures without insert fragments. See, Aslandis anddejong, Nucleic Acids Research 18:6069-6074 (1990). Examples ofpreferred flanking sequences for LIC are: “LIC-F”:5′-GGTATTGAGGGTCGCATC-3′ (SEQ ID NO: 2) and “LIC-R”:5′-AGAGGAGAGTTAGAGCCATC-3′ (SEQ ID NO: 3) in biotinylated andnon-biotinylated forms (HPLC purified, QIAGEN Operon, Alameda, Calif.).

III. Optimization of Method for High-Throughput Selection

A. Interacting the Target with the Aptamer Library

In the initial round of selection, the library should be incubated withthe bead-bound target to produce bead-target-aptamer sequence complexes.In this step it is helpful to use at least a 10-fold molar excess ofssDNA in a volume to a 10 nM concentration of the target molecule,however, it may not be necessary. A preferred solution and protocol forinteracting the library with the bead-bound target is the following: 1nmol of the library is diluted into 100 μL of a buffer in a tube andheated to 95° C. for 2 min then immediately cooled at 4° C. Thismaterial was added to 10 mL of PBS-T containing 1 μg/mL BSA, 0.1 μg/mLdIdC. 100 pmol of bead-bound target is then added to this mixture andincubated with rotation for 30 min at room temperature.

B. Stringency of Interaction Conditions

The interaction conditions will affect the affinity of the aptamersselected. Therefore, the aptamer library should interact with the targetmolecule at a concentration preferably at or below the desired affinity.For example, if affinity of 10⁻⁶ M or higher is desired, theconcentration of the target molecule is preferably at or below 10⁻⁶ M.The selection stringency can be adjusted by choosing the appropriatebinding and washing conditions as is known in the art. For example, theselection stringency can be manipulated with salt concentrations between50 mM and 250 mM NaCl or preferably between 100 mM and 200 mM NaCl. Theinventions also contemplates that the selection stringency can beadjusted by varying the amount of binding time, wash volumes, magnesiumor cation concentrations, or the concentration of target or librarysequences present.

After allowing the aptamer library to interact with the magnetic beadbound-target molecule preferably as mixture in a vessel, magnetic forceis applied to the mixture to separate bead-target-aptamer sequencecomplexes from unbound aptamer sequences.

C. Releasing and Amplifying Selected Aptamers

In one embodiment, the mixture in the vessel is subject to a magneticfield and magnetic beads are retained on the side of the tube whileunbound aptamers can be washed away with buffer. The washing procedureis repeated several times under either the same or more stringentconditions applied to the bead-target-aptamer sequence complexes toselect for higher affinity aptamers. Once the supernatant is removed,the bead-target-aptamer sequence complexes and magnetic beads can bewashed with buffer. The aptamers bound to the target can be eluted andthen subjected to PCR amplification using primers that are complementaryto the flanking ends of the aptamer sequence. Elution is preferably doneby resuspending the bead-target-aptamer sequence complexes in buffercompositions. For example, 10 ul 20 mM Tris, 500 mM imidazole, pH 7.5.

PCR amplification of the aptamer sequence is preferably performed usinga forward primer and a biotinylated reverse primer conplementary to theflanking ends to facilitate separation of the DNA strands afteramplification. For example, 1 μM primers “LIC-F” (SEQ ID NO: 2) andbiotinylated “LIC-R” (SEQ ID NO: 3) were used in Example 1. Using theseprimers will generate PCR products with a biotin moiety attached to thenon-aptamer strand. Single-stranded aptamers (non-biotinylated strand)are then separated from the complementary strand and addition of amagnet to capture the biotinylated non-aptamer strand. Separation of thesingle-stranded aptamers from the complimentary strand can be carriedout by incubation with heat, or more preferably, incubation with astrong base, such as NaOH. After separation, the ssDNA aptamers areremoved in the supernatant and diluted. Finally, the single-strandedaptamers are heated for several minutes then immediately placed at 4° C.until the next round of selection.

In order to remove aptamers that bind to the magnetic beads and not thetarget, counter-selection should be performed after several rounds.Counter-selection is done without any target present to remove anyaptamers that might bind to the beads alone.

D. Optimization of Selection Cycles

In a preferred embodiment, the enrichment of aptamers that bind aparticular protein of choice should be performed for a sufficient numberof cycles. The enrichment of the aptamers should be monitored after atleast 5, 10, and 15 rounds of selection for enrichment. Satisfactoryenrichment should be accomplished within 10 to 15 cycles of selection(See FIGS. 6 and 8).

For additional rounds of selection, the amount of protein is recommendedto be reduced by about half, in rounds 2-10, and subsequently in halfagain for rounds 11-15, and so on. Reduction in the incubation time isalso recommended. After round 2, the PCR cycle number should also bereduced, preferably to 10 cycles, because of the increased likelihood ofamplification of products of incorrect size. More than 15 cycles ofamplification may lead to the production of larger fragments, and lateridentified as concatamers.

In one embodiment, 15 rounds of selection should produce high affinityaptamers using manual or high-throughput processing. The throughput ofthe present method is approximately 32 aptamers per month and amenableto a multi-well high throughput approach which may be scaled-up toproduce about 384 aptamers per month.

IV. Further Amplification of Isolated Aptamers

The enriched aptamer sequence that binds to the target molecule ofinterest can be cloned into an appropriate vector for furtheramplification and sequencing.

After sufficient number of rounds to complete enrichment, thesingle-stranded aptamer sequences should be amplified by PCR and the PCRproducts concentrated and purified. In one embodiment, the PCR primersequences are primers complementary to the flanking sequences in theaptamer sequences. The purified aptamer sequences can then be clonedinto a vector, and transformed into E. coli. A sufficient number ofcolonies are picked for each sample, and the isolated plasmids areamplified and then sequenced.

Exemplary plasmids and vectors that can be used for cloning include, butare not limited to, LIC vectors such as pUC18LIC and pET30XaLIC.

The amplification of the cloned aptamer sequence before sequencing canbe done by rolling circle amplification or by traditional concentrationand purification using such commercially available kits as a 96-wellmini-prep (Qiagen, Valencia, Calif.). However, the rolling circleamplification system may be more preferred in some cases because it iseasier, more efficient and cost-effective. The cloned aptamer sequencecan be then sequenced by known sequencing means such as using a T7promoter primer in the Big-dye Terminator kit (Qiagen, Valencia, Calif.)and run on a sequencer.

Sequences can be analyzed and aligned using such programs as CLUSTALX v.1.81 (Higgins D G, Sharp P M. (1988) Gene December 15; 73(1):237-44)) orother alignment programs. Pattern analysis is suggested to be performedusing a program such as CONSENSUS (G. Z. Hertz and G. D. Stormo. (1995)Proceedings of the Third International Conference on Bioinformatics andGenome Research 201-216).

V. Evaluating Isolated Aptamers for Binding Activity

The term “binding activity” is herein meant to describe the measure ofthe strength of the binding or affinity of molecules to each other. Thefunctional binding activity of aptamers selected by the method of theinvention can be illustrated in several assays. These assays shoulddetermine the dissociation constant (K_(D)), which is a measure of thestrength of binding activity or affinity between the isolated aptamersand the target molecules. As is known in the art, a low dissociationconstant indicates stronger binding and affinity of the molecules toeach other. Therefore, a preferred dissociation constant should be atleast 10⁻⁴M, more preferably at least 10⁻⁶M, most preferably at least10⁻⁸ and 10⁻⁹M.

Enzyme-linked assays provide a means of quickly evaluating a group ofaptamers from a selection by measuring their relative affinities, andthis kind of triage can be used to prioritize aptamers for more detailedcharacterization See Drolet D W, Moon-McDermott L, Romig T S. (1996) Anenzyme-linked oligonucleotide assay. Nat. Biotechnol. August;14(8):1021-5, which describes the assay and is hereby incorporated byreference. Enzyme-linked assays can also provide information aboutcross-reactivity. Enzyme-linked assays offer advantages over othertechniques, such as equilibrium dialysis and electrophoretic-mobilityshift assays that are conventionally used to evaluate aptamers from aselection. These advantages include the lack of radioisotope usage,increased throughput in a multi-well plate, minimization of waste, andease of precise quantitation of the relative binding affinities.

Using aptamers in a protein blot analysis is another means ofcharacterizing their specificity. For example, the TTF1 Aptamers A and Cselected for in Example 1 were tested by chemiluminescent protein blotanalysis, however only the TTF1 AptamerA worked in this applicationsuggesting that, just as some antibodies fail to recognize the denaturedform of a protein, some aptamers will recognize epitopes that are absentin the denatured form of the protein. The TTF1 aptamer showed nocross-reactivity to E. coli proteins in a cleared lysate on the blot andwas similar in specificity observed for the anti-PentaHis-HRP antibody.

Lastly, the binding activity of the aptamers of the invention can beanalyzed through surface plasmon resonance (SPR). The SPR technique isan optical method for measuring the refractive index of materialsadsorbed on a metal, such as the difference between the refractive indexof a buffer (i.e. water) and the refractive index of a molecule bound tothe surface. Examples of SPR platforms to perform SPR include includebut are not limited to those available from Applied Biosystems (FosterCity, Calif.) and Biacore Inc. (Piscataway, N.J.).

VI. Utility of Selected Aptamers

The aptamers isolated by the methods described by the invention can beused as affinity ligands to separate and purify target molecules, asprobes to trace, monitor, detect and quantitate target molecules, or toblock, allow, activate or catalyze reactions that are physiologicallyrelevant to achieve therapeutic effect. Aptamers so isolated haveutilities similar to antibodies. They can act as a pharmaceutical agent,bind to a specific target and direct specific molecules to a desiredsite, and/or they can inhibit or promote a physiologically relevantreaction to achieve a desired therapeutic effect. Various in vivo, exvivo, and in vitro methods can employ aptamers isolated by methodsdescribed herein, as will appreciated by one of skill in the art.

Within respect to in vitro procedures, aptamers can be used in affinitypurification matrixes to purify target molecules. The subject aptamersare ideal for chromatographic separations of target molecules fromcontaminants and for purifying target protein molecules from cellcultures or cell extracts. The immediate application of these aptamersis to purify antibodies, enzymes, hormones, receptors, and factors thatare used in research, development, diagnostic, pharmaceutical, industryapplications.

In one embodiment, the key function of high affinity aptamers inapplications such as protein purification, protein profiling chips,surface plasmon resonance and diagnostics is to recognize and separatethe target protein from a complex mixture of proteins.

Also described herein is the successful application of aptamer affinitychromatography for one-step purification of a protein from the complexmixture of proteins in the soluble fraction of bacterial cell lysates.Although aptamer affinity chromatography has been described anddemonstrated for the purification of a protein from conditioned cellculture media, this purification technique has not been previouslydemonstrated for more complex samples such as cell cultures and serum.Detrimental effects from DNase activity in purification from bacteriallysates were not observed using the aptamers isolated by the methoddescribed herein, unlike the problems associated with DNase degradationof aptamers that occurs when purifying targets from serum (Romig T S,Bell C, Drolet D W. (1999) Aptamer affinity chromatography:combinatorial chemistry applied to protein purification. J Chromatogr BBiomed Sci Appl. August 20; 731(2):275-84). Importantly, aptameraffinity chromatography provides a means of protein purification of thenative form of a protein without relying on affinity tags that mayadversely affect protein structure, function or ability to form crystalsfor structural characterization.

Selected aptamers having specific chiral properties can be used toseparate chiral compounds and obtain optically pure chemicals. They canalso be used in place of antibodies in various research, development anddiagnostic applications such as blotting techniques, flow cytometry,immunoassays, strip assays, immunohistological techniques, affinitysensors, etc. Aptamers selected by this methods can further be used tomonitor, trace, detect and quantitative desired target such as proteins,antibodies, microbes, virus, bacteria, macromolecules, and smallmolecules and used as valuable tools for proteomics studies of proteinand their function.

In addition, the Examples fully demonstrate the abilities of aptamersgenerated by the method to bind their target protein with high affinityand specificity, and detail their uses in a number of assays. It is alsofurther contemplated that one could use a pool of aptamers in a givenapplication; for example, a mixture of 6H5 (SEQ ID NO: 14), 6H1 (SEQ IDNO: 15) and/or 6H7 (SEQ ID NO: 16).

Herein described are a series of aptamers which bind to a polyhistidinesequence coupled to various proteins and used in many applicationsincluding as protein purification reagents. These His6 aptamers isolatedare useful for the capture and detection of target molecules thatcontain or are fused to polyhistidine residues (His6). The use of a His6tag for identification, detection, purification and manipulation ofproteins and other target molecules is well known in the art. Therefore,aptamers that bind or detect His6 tags may be useful in many biologicalapplications including, but not limited to, affinity resins, magnetic orpolymer beads, as a diagnostic detection reagent, to capture orimmobilize reagents for diagnostic, detection or quantitative studies.

In a preferred embodiment, the aptamer sequences used to bind to His6affinity tags have at least 80% homology, more preferably 90%, and morepreferably at least 95% homology to the degenerate aptamer sequences ofSEQ ID NOS: 14, 15 and 16. The present aptamers can be flanked by primersequences, such as the primers of SEQ ID NO: 17 and SEQ ID NO: 18 asdescribed herein. It is contemplated that these aptamer sequences may bevaried in the degenerate sequence region, by up to 20 percent, morepreferably by 10 percent, most preferably by 5 percent, while retainingthe functional properties as described herein.

One aspect of the present His6 aptamers is their application to solidsupports. In one aspect, arrays of tagged proteins can be immobilized inan array on a solid support, in which the solid support has been spottedwith an aptamer such as the aptamers of SEQ ID NOS: 14, 15 and 16. Inanother aspect, solid supports used in SPR may be modified to accept thepresent aptamers, and his-tagged proteins used as target molecules to becaptured or immobilized by the present aptamers.

In one embodiment, the aptamer can be used as a capture agent to bind orimmobilize a target protein to a solid support. The solid support can beany comprised of substrates having the structure and compositioncommonly associated with filters, wafers, wafer chips, membranes andthin films that one of ordinary skill in the art is aware of. However,it is contemplated that the solid support may be comprised of substratesincluding but not limited to resins, affinity resins, magnetic orpolymer beads, or any a diagnostic detection reagent, to capture orimmobilize reagents for diagnostic, detection or quantitative studies.

Further the solid supports may comprise any material depending on thedesired use, including but not limited to glass, metal surfaces andmaterials such as steel, gold, silver, aluminum, copper, silicon, andglass, ceramic or polymeric materials such as polyethylene,polypropylene, polyamide, and polyvinylidenefluoride, etc. andcombinations thereof. The solid support can be coated with any materialto facilitate the attachment, binding, hybridization, immobilization orinteraction of biological molecules on the surface. In one embodiment,the solid support is a glass slide having a layer of gold,carboxymethylation, dextran, collagen, avidin or streptavidin to supportthe attachment or interaction. A variety of molecules can then beattached to the solid support by means of this attachment orinteraction. For example, if the support is coated with streptavidin,biotinylated oligonucleotides can be attached to the support by means ofthe strong interaction between biotin and streptavidin.

In this manner, the His6 aptamers of the invention can then be attachedto the surface of the support through the layer of material coating thesolid support. The term “attached to said solid support” herein is meantattached, removeably attached, bound, covalently bound, non-covalentlybound, conjugated, hybridized, immobilized or interaction with thesupport surface; there may be any number of intervening layers ormolecules between the aptamer and the solid support, that one ofordinary skill in the art will recognize depending on the intended use.For example, the aptamer can be attached to the support by means of anoligonucleotide that is covalently attached to the support on one endand then attached to the aptamer through base complementarity on theother end of the oligonucleotide.

It is also contemplated that arrays of the aptamers are attached to thesolid supports. It is understood that the array may be as few as 2aptamers or as many as 10,000,000 depending on the desired end use. Itis further contemplated that arrays of the aptamers can be used tocapture large amounts of a molecule or protein of interest from asample. The captured and immobilized molecules will permit diagnostic,biological, qualitative and quantitative studies requiring immobilizedor bound proteins, ligands, and peptides.

In one embodiment, as shown in FIG. 12B, the solid support isstreptavidin-coated having a biotinylated His6 aptamer selected from SEQID NOS: 14, 15 and/or 16, attached to the support, thereby providing asurface that can capture and bind to any His6 tagged protein, ligand orpeptide. The resulting support having immobilized His6 tagged proteins,ligands or peptides will permit diagnostic, biological, qualitative andquantitative studies requiring immobilized or bound target molecules.Furthermore, the His6 aptamer does not easily disassociate from solidsupport but remains firmly attached as shown in the Examples. Therefore,the immobilized target molecules should not be easily dissociated fromthe solid support upon binding the aptamer.

In another embodiment, as shown in FIG. 12C, the solid support isstreptavidin-coated and attached to the support is a biotinylatedoligonucleotide that binds to a fixed portion of at least one of theHis6 aptamers of SEQ ID NOS: 14-16 by base complementarity. One withordinary skill in the art would recognize how to design such anoligonucleotide using basic complementary nucleic acid bases, i.e. A-Tand C-G. This would also provide a surface that can capture and bind toany His6 tagged proteins and peptides applied to the surface. Theresulting support will permit diagnostic, biological, qualitative andquantitative studies requiring immobilized or bound proteins, ligands,and peptides. The added flexibility in this embodiment, is that the chipcan be used with any His6 aptamer of the invention as well as theaptamers isolated using the methods described herein.

Supports having the aptamers attached thereto as described above arestable and can be stored for sufficiently longer periods of time thancurrent available supports using antibodies as capture reagents becauseof the longer shelf-life of DNA and nucleic acids. As shown later in theExamples, the aptamers have equal or better ability to captureHis6-tagged proteins, peptides or ligands. Furthermore, the aptamer orthe proteins and ligands bound, can be removed from the solid supportallowing the solid support to be reused with different binding pairs.

Example 1 Library Creation and Aptamer Selection

An improved protocol for DNA aptamer production that is relatively easyand scalable without the need for expensive robotics is describedherein. Thyroid transcription factor 1 (TTF1), a well characterizedmember of the NK homeodomain transcription factors (See Harvey R P.(1996) NK-2 Homeobox Genes and Heart Development. Developmental Biology.178, 203-216; and Ristoratore F, Spagnuolo A, Aniello F, Branno M,Fabbrini F, Di Lauro R. (1999) Expression and functional analysis ofCititf1, an ascidian NK-2 class gene, suggest its role in endodermdevelopment. Development. November; 126(22):5149-59) was used as atarget molecule. TTF1 is expressed in the developing thyroid, lung, andbrain of vertebrates, and several effector genes have been identified inthyroid and lung tissues. The DNA recognition site of TTF1 differs fromother homeodomain containing proteins, attributed to the NK-typehomeodomain (Guazzi S, Price M, De Felice M, Damante G, Mattei M G, DiLauro R. (1990) Thyroid nuclear factor 1 (TTF-1) contains a homeodomainand displays a novel DNA binding specificity. EMBO J November;9(11):3631-9). Following 15 rounds of selection, the affinity andspecificity of several aptamers were characterized, and their uses inassays were described for the capture and identification of proteins,such as Western blots, enzyme-linked assays, and affinity purification.

Cloning, protein expression, and purification. Thyroid transcriptionfactor 1 (TTF1) was cloned from Ciona intestinalis 16 hour embryosfollowing RNA isolation (Trizol reagent, Gibco, Carlsbad, Calif.) andfirst strand synthesis (Superscript First Strand Synthesis System forRTPCR, Gibco, Carlsbad, Calif.) by PCR using the following gene specificprimers that contained 5′ Ligation Independent Cloning (LIC) (Novagen,Madison, Wis.) compatible ends: Forward5′-GGTATTGAGGGTCGCTCAGTTAGCCCAAAGCATTCG-3′ (SEQ ID NO: 19); Reverse5′-AGAGGAGAGTTAGAGCCTTATCGGTAAACACTGTACAGGATCG-3′ (SEQ ID NO: 20).

LIC was performed as previously described in Doyle S A, Murphy M B,Massi J M, and Richardson P M (2002) High-Throughput Proteomics: AFlexible and Efficient Pipeline for Protein Production. J. ProteomeResearch December; 1(6):531-536), to insert the coding sequence of Cionaintestinalis TTF1 into the vector pNHis, which adds a hexahistidine tagto the amino-terminus of the encoded protein.

Subcloning. An expression vector was constructed from pET30 (Novagen,Madison, Wis.) by removing 117 bp 3′ of the hexahistidine tag site thatencoded extra affinity tags and by adding the sequence5′TCCGGTATTGAGGGTCGCTCTAACTCTCCTCTG 3′ (SEQ ID NO: 4) to allow for LICcloning (FIG. 1). Construction of the LIC vector containing theN-terminal hexahistidine affinity tag is shown in FIG. 1. The new vectorpNHis, encodes a protein with a N-terminal extension of 6 histidineresidues followed by 6 additional amino acids that encode a factor Xacleavage site. A stop codon was added to the gene sequence so that the3′ LIC sequence did not add 6 extra amino acids to the C-terminus of theprotein sequence.

The new vector, pNHis, was linearized within the LIC sequence bydigestion with BseR1, treated with mung bean nuclease to produce bluntends, and gel purified. Cloning was performed as described in LICcloning manuals (Novagen, Madison, Wis.). Briefly, the gene sequenceswere amplified by PCR using sequence specific primers with 5′ adaptors(forward primer: 5′ GGTATTGAGGGTCGC (SEQ ID NO: 5) and reverse primer:5′ AGAGGAGAGTTAGAGCCTTA (SEQ ID NO: 6)). The insert and vector DNA weretreated with T4DNA polymerase in the presence of dGTP and dCTP,respectively, for 40 min. at room temperature (22° C.), and thepolymerase was heat inactivated for 20 min. at 75° C. The fragments wereannealed in a 10 min. reaction at room temperature, and transformed intoNOVABLUE competent cells (Novagen, Madison, Wis.). Positive clones wereconfirmed by a colony PCR procedure using the T7 promoter (5′TAATACGACTCACTATAGGG 3′ (SEQ ID NO: 7)) and T7 terminator (5′GCTAGTTATTGCTCAGCGG 3′ (SEQ ID NO: 8)) primers, and confirmed bysequencing.

Two sources of coding sequence were used, the bacterium Xylellafastidiosa, and Ciona intestinalis, a primitive chordate. Eight randomlychosen Xylella proteins, ranging from 10 to 32 kDa were cloned into theexpression vector. Five full-length Ciona proteins (42 to 88 kDa) werealso cloned into this vector, as well as several gene fragmentscontaining DNA binding domains.

Expression Screening. Expression constructs from Xylella fastidiosa andCiona intestinalis were tested under various growth conditions using thedot blot procedure to identify optimal growth conditions for eachprotein (FIG. 2). Expression screen dot blots of Xylella and Cionasamples, grown for 4 hours or overnight (o/n) were analyzed to determinewhich samples should be chosen for further purification. The standardcurve generated on each blot was reproducible and used to approximatethe concentrations of protein in the sample spots. A good correlationbetween spot intensities and protein concentration was obtained (datanot shown).

The High-throughput Protein Purification (96-well) protocol used topurify the proteins can be described briefly as follows: (1) Grow andlyse cultures in 24-well blocks, then pellet insoluble material bycentrifugation. (2) Incubate clear lysate with Ni-NTA resin in batch.Combine 50 μl of 50% slurry Ni-NTA Superflow (Qiagen, Valencia, Calif.)with cleared lysate in 96-well filter plate (Genemate) and mix at 4° C.for 20 min. (3) Wash wells 3× with 750 μl wash buffer on vacuummanifold. (4) Elute proteins into 96-well microtiter plate on vacuummanifold. Add 100 μl elution buffer (containing imidazole and 10%glycerol), incubate 5 min, and apply vacuum.

Plasmids encoding Xylella genes were transformed into BL21 (DE3) Goldcells (Stratagene, La Jolla, Calif.) and plasmids encoding Ciona geneswere transformed into Rosetta pLysS cells (Novagen, Madison, Wis.) basedon previous experiments. Initial starter cultures grown at 37° C. wereused to inoculate 5 ml LB medium containing 50 μg/ml kanamycin in24-well blocks. Once an O.D₆₀₀ of 0.6 to 0.8 was reached, the cultureswere induced with IPTG at a concentration of 0.1 mM or 1.0 mM, and grownat various temperatures (18° C., 25° C., 30° C. and 37° C.) for 4 hoursor overnight. In the present study only temperature, and inductionstrength and time were tested, however, many other conditions (growthmedium, host cells) can be added to the screen.

The cells were harvested by centrifugation, frozen at −70° C., thenthawed and resuspended in 0.5 ml lysis buffer (50 mM NaH₂PO₄, 300 mMNaCl, 2 mM MgCl₂, 20 mM imidazole, pH 8.0) containing 1 mg/ml lysozyme,0.5 μl (12U) Benzonase nuclease (Novagen, Madison, Wis.), and 2 μlprotease inhibitor cocktail (Sigma, St. Louis, Mo.). Followingincubation on a plate shaker at 4° C. for 30 min, an aliquot of crudelysate was removed, and the remainder of the sample was clarified bycentrifugation.

Two μl of crude and cleared lysate were spotted on Protrannitrocellulose membrane (Schleicher and Schuell, Keene, N.H.) using a12-channel pipette. A serial dilution of protein standard was alsospotted (15-1500 ng of a 42 kDa protein). The membrane was incubatedusing the Western Processor developing system (Biorad, Hercules, Calif.)as follows: TBS (6 mM Tris-Cl, 150 mM NaCl, pH 7.5), 5 min, 3 cycles;blocking buffer (TBS with 3% BSA) 30 min; TBS T/T (TBS with 0.05% Tween20 and 0.2% Tritin X-100), 5 min., 3 cycles; PentaHis HRP conjugate(Qiagen, Valencia, Calif.) (1:1000 in blocking buffer) 30 min.; TBS T/T,5 min., 5 cycles. The membrane was then treated with metal-enhanced DABsubstrate (Pierce) following the manufacturer's protocol, and scannedusing a flatbed scanner. The blotting procedure was completed in lessthan 2 hours.

Overall, the Xylella fastidiosa proteins showed good total expressionlevels, with nearly all of the crude lysate samples exhibiting highintensity spots (data not shown). Two samples (proteins XF0233 andXF2614) showed little difference in the total protein and solubleprotein samples under all conditions tested, indicating that theseproteins are very soluble. For the remainder of the samples, however, atrend was seen where the solubility was increased with reduced growthtemperature (≦25° C.). In addition, overnight induction conditionsproduced better protein yields than 4 hour induction conditions. Nearlyidentical results were obtained when samples were induced with 0.1 and1.0 mM IPTG (data not shown).

The total expression levels for the Ciona intestinalis samples were notas consistent as in the Xylella set. Unlike the Xylella proteins, totalexpression was better in samples induced for 4 hours than overnight,although not at every temperature tested. Snail and TTF-1 expressedsoluble protein under most conditions, while the remainder of theproteins showed reduced solubility when induced overnight. In the 4 hoursamples, snail and TTF-1 produced their highest soluble yields at ≧25°C., where 25, 30 and 37° C. were roughly equivalent. For the proteinsshowing lower expression levels, no clear pattern was seen. Hox1produced more soluble protein at ≧25° C., with the highest yield at 37°C., while Tbx2/3 and Tbx6 produced the highest yields of soluble proteinat 30° C. These differences are probably due to the inherent stabilityof these proteins when expressed in bacteria. As with the Xylellasamples, nearly identical results were obtained when the sameexperiments were performed with 0.1 and 1.0 mM IPTG induction (data notshown).

Significant differences were seen in the expression of full-length andDNA binding domains of Ciona proteins Tbx6 and Tbx2/3. The domain ofTbx6 showed little or no expression under any conditions, unlike thefull-length protein, which expressed fairly well at higher temperaturefor 4 hours. The TBX2/3 domain (Tbx2/3d) clearly showed highest yieldsat ≦25° C., while the full-length protein expressed more soluble proteinat ≧25° C. These results suggested that the cloned DNA fragments do notcontain structurally stable DNA binding domains, and thus the smallerprotein products were less stable than the full-length proteins.Comparison of additional full-length proteins and fragments will provideuseful information regarding the ability of protein fragments containingspecific domain motifs to fold into stable protein products whenexpressed in bacteria.

This expression screen allows for the identification of optimalconditions for soluble protein expression in a convenient andreproducible manner. The use of 24-well blocks in standard incubatorsfor cell growth, standard lysis procedures, and simple centrifugationand sample spotting steps make it easily implemented, without the needfor expensive robotics. In many cases, protein expression levels aresufficiently high to allow for direct purification of micrograms ofprotein from 5 or 10 ml cultures grown in the 24-well blocks.Alternatively, if more protein is required, the optimal conditions forscaled up experiments can be identified using this method.

After screening several expression conditions, TTF1 was found to be mosthighly expressed in Rosetta (DE3) pLysS cells (Novagen, Madison, Wis.)induced with 1.0 mM IPTG at 37° C. for 4 hours. One liter of culturegrown under these conditions was harvested by centrifugation at 5,000×gfor 10 min at 4° C. The pellet was resuspended in 30 mL of resuspensionbuffer (50 mM Na₂HPO₄, 300 mM NaCl, 20 mM imidazole, 0.1% Triton X100, 5mM 2-mercaptoethanol at pH 7.0 with 1 mM PMSF Plus(Roche-Applied-Science, Indianapolis, Ind.), and 1× Protease InhibitorCocktail (Sigma Chemicals, St. Louis, Mo.)). The cells were then lysedwith the Emulsiflex-C5 (Avestin, Ottawa, Ontario, Canada) homogenizer at15,000 psi. The lysate was centrifuged at 15,000×g for 25 min at 4° C.to remove insoluble material. Chelating Sepharose High Performance resin(Amersham Biosciences, Piscataway, N.J.) was charged with 0.1 M NiSO₄and washed with 10 column volumes of sterile water. The cleared lysatewas incubated with 300 μL of 50% slurry of Ni⁺² charged resin and boundin batch for 20 min with constant rotation then loaded onto an emptypolypropylene column (Qiagen, Valencia, Calif.) and allowed to drain bygravity flow. The 150 μL column was then washed with 50 column volumesof resuspension buffer containing 20% glycerol and the bound proteinswere eluted with 0.5 column volumes of elution buffer (1 M imidazole, 50mM Na₂HPO₄, pH 7.0, 150 mM NaCl, 0.1% Triton-X100, 20% glycerol, and 5mM 2-mercaptoethanol).

Determination of Protein Yield and Purity. The concentration and puritywere determined using 4 μL in a Protein 200 LABCHIP kit (Caliper,Newton, Mass.) run on an Agilent 2100 Bioanalyzer (Agilent Technologies,Palo Alto, Calif.). The protein LABCHIP was prepared by injecting 12 μlof a gel matrix and fluorescent dye mixture into the chip using a chippriming station. The samples were prepared by mixing 4 μl of protein and2 μl of a SDS-based denaturing sample buffer containingβ-mercaptoethanol as well as an upper and lower mass standard and byboiling the mixture. Samples and ladder were then diluted to 90 μl withwater and 6 μl of each diluted sample was loaded into a well of theLABCHIP. Dilution of the samples is necessary to decrease backgroundfluorescence due to the SDS in the sample buffer. The LABCHIP was thenplaced in an Agilent 2100 Bioanalyzer, and electrophoresed for 30minutes. Agilent Biosizing software was used to determine the size ofthe proteins of interest by normalization against two internal standardsof 6 and 210 kDa. The fluorescent peak identification settings wereadjusted for sensitivity, 0.8 for the minimum peak height, 0.2 secondsfor the minimum peak width, and 4 for the slope threshold.

In vitro selection of aptamers. A degenerate oligonucleotide library wassynthesized at 1 μmole scale and HPLC purified (QIAGEN Operon, Alameda,Calif.). This material was diluted to 0.1 nmol/μL in 10 mM Tris, pH 8,and stored at −20° C. This library, referred to in this example as“LIC-Apt”, is composed of 40 random nucleotides flanked by sequencessuitable for ligation independent cloning (LIC):5′-GGTATTGAGGGTCGCATC-3′ (SEQ ID NO: 17) and 5′-GATGGCTCTAACTCTCCTCT-3′(SEQ ID NO: 18).

Primers that anneal to the 5′ and 3′ sequences flanking the degenerateregion of LIC-Apt that were used during the selection and cloning were:“LIC-F”: 5′-GGTATTGAGGGTCGCATC-3′ (SEQ ID NO: 2); “LIC-R”:5′-AGAGGAGAGTTAGAGCCATC-3′ (SEQ ID NO: 3); in biotinylated andnon-biotinylated forms (HPLC purified, QIAGEN Operon, Alameda, Calif.).

Protein-bound Ni-NTA magnetic beads were prepared by first equilibrating150 μL of a 5% slurry (approximately 45 μg capacity) of Ni-NTA magneticbeads (Qiagen, Valencia, Calif.) into PBS-T (50 mM K₂HPO₄, pH 7.5, 150mM NaCl, 0.05% Tween 20). The equilibrated beads were resuspended in1250 μL of PBS-T and 25 μL of 2 mg/mL purified TTF1 was added (a 1:50dilution to lower the imidazole concentration) and mixed with rotationfor 30 min at 4° C. The bead-bound TTF1 was then washed 3× with 1 mLPBS-T, and diluted to 0.25 μg/μL (5 pmol/μL of 50 kDa TTF1) with PBS-Tand stored at 4° C.

In the initial round of selection, the “LIC-Apt” library was incubatedwith the bead-bound TTF1 using a 10-fold molar excess of ssDNA in avolume that gave a 10 nM TTF1 concentration. 1 nmol of “LIC-Apt” wasdiluted into 100 μL of PBS-T in a PCR tube and heated to 95° C. for 2min then immediately cooled at 4° C. This material was added to 10 mL ofPBS-T containing 1 μg/mL BSA, 0.1 μg/mL dIdC. 100 pmol of bead-boundTTF1 was then added to this mixture and incubated with rotation for 30min at room temperature. The tubes were then applied to a magnet (DexterMagnetics, Elk Grove Village, Ill.), the supernatant removed, and thebeads were washed 3× with 1 mL PBS-T, mixing by inversion for each washstep. The proteins and bound aptamers were eluted from the Ni-NTAmagnetic beads with 10 μL of 20 mM Tris, pH 7.5, 500 mM imidazole andtransferred to PCR tubes. 100 μL PCR reactions contained 1.25 units Pfxpolymerase (Invitrogen, Carlsbad, Calif.), 1 μM primers “LIC-F” andbiotinylated “LIC-R”, 0.1 mM dNTPs, 0.5 mM MgSO₄, and 0.1× enhancersolution. Amplification conditions were 2 min at 95° C.; 15 cycles: 30sec at 95° C.; 30 sec at 56° C.; 30 sec at 68° C.; 2 min at 68° C. Thisprotocol produced 2-5 μg of the correct size product as determined usinga DNA 500 lab-chip (Caliper, Newton, Mass.) on an Agilent 2100Bioanalyzer. After the amplification step, 90 μL of the PCR product and23 μL 5M NaCl were then mixed with 1 mg of M-280 streptavidin magneticbeads (Dynal Biotech, Brown Deer, Wis.) for 10 min at room temperature,then washed 3×1 mL with PBS-T. Single-stranded aptamers(non-biotinylated strand) were separated from the immobilizedcomplementary strand using a 5 min incubation of 50 μL of fresh 100 mMNaOH. The tubes were applied to a magnet and the ssDNA was removed anddiluted into 1 mL PBS-T, containing 10 μL of 100 mM monobasic phosphatebuffer to adjust the pH to 7.5. Finally, the material was heated to 95°C. for 2 min then immediately placed at 4° C. until the next round ofselection.

For additional rounds of selection, the amount of protein was reduced to50 pmol (rounds 2-10), and subsequently 25 pmol (rounds 11-15) in abinding volume of 1 mL, and the incubation time was reduced to 10 min.After round 2, the PCR cycle number was reduced to 10 cycles because ofthe amplification of products of incorrect size. More than 15 cycles ofamplification often led to the production of larger fragments, lateridentified as concatamers. In order to remove aptamers that bind to theNi-NTA magnetic beads, counter-selection was performed after rounds 3,6, 9, and 12. A 20 μL aliquot of a 5% slurry of Ni-NTA-magnetic beadswas added to the 1 mL of ssDNA in PBS-T and incubated for 10 min withrotation, then applied to a magnet and the supernatant removed for thenext round.

After round 15, the material was amplified by PCR with “LIC-F” and“LIC-R” primers, and the products were purified with MinElute (Qiagen,Valencia, Calif.), LIC-cloned into pET30XaLIC vector (Novagen, Madison,Wis.), and transformed into NovaBlue E. coli (Novagen, Madison, Wis.).32 colonies were picked for each sample, and the plasmids purified by96-well mini-prep (Qiagen, Valencia, Calif.). The plasmids weresequenced using a T7 promoter primer in the BIG_DYE Terminator kit andrun on an ABI 3730 Sequencer (Applied Biosystems, Foster City, Calif.).Sequences were aligned using ClustalX v. 1.81 (Higgins D G, Sharp P M.(1988) CLUSTAL: a package for performing multiple sequence alignment ona microcomputer. Gene December 15; 73(1):237-44). Pattern analysis wasperformed using CONSENSUS (G. Z. Hertz and G. D. Stormo. (1995)Identification of consensus patterns in unaligned DNA and proteinsequences: a large-deviation statistical basis for penalizing gaps.Proceedings of the Third International Conference on Bioinformatics andGenome Research 201-216).

Five groups of identical sequences were identified after the 15 roundsof selection, including one aptamer “A” that represented 30% of thetotal evaluated. Thus, the selection conditions using magnetic beadswere sufficiently stringent for successful enrichment in 15 rounds. Thedegenerate portions of the aptamers isolated are listed as follows:

TTF1 Aptamer A SEQ ID NO: 9 TCAAAAGGGGTGATTGCTTGCACAATGACAGGGTAGGACATTF1 Aptamer B SEQ ID NO: 10 GATACACGGGCGGAGGAGGTGGGGGGGGGTAGGTGGGTATTTF1 Aptamer C SEQ ID NO: 11 TGGCTAGTGGGTAAGGGGCGGGAGGGTGACAGGGCGATCCTTF1 Aptamer D SEQ ID NO: 12 TTATGGGGATGAAAGTGGTGTTCGGGTTCGCCACTTCCACTTF1 Aptamer E SEQ ID NO: 13 TTGGGGTGGGAGGGCGGGTTAACAAAGATAGCGCAACAGG

The aptamers that were generated (FIG. 4) did not have affinity towardsthe Ni-NTA magnetic beads (not shown). Interestingly, there were 2aptamers—TTF1 AptamerA and TTF1 AptamerC—that contained a consensussequence (FIG. 4), although this consensus may not necessarily beresponsible for the affinity towards TTF1 since all the aptamersequences obtained displayed affinity towards the protein (not shown).In addition, several G repeats were found in each group. The TTF1 dsDNAbinding consensus sequence (5′ T(C/T)AAGTG 3′) is not contained in anyof the aptamer sequences. In addition to the aptamers described in FIG.4, there were 3 sequences that were each represented as singletons (notshown).

Example 2 Determination of the Specificity of a TTF1 Aptamer

We used an enzyme-linked assay in order to prioritize the aptamers fromthe TTF1 selection for further characterization (not shown). This assayprovided a rapid assessment of the relative binding capabilities of manyaptamers from a particular selection experiment. The results suggestedthat TTF1 AptamerA may have the highest affinity for TTF1 (laterconfirmed by kinetic studies) and thus was chosen for furthercharacterization. In addition, the enzyme-linked assay was used toprovide information regarding cross-reactivity (FIG. 5). Employing acalorimetric detection system (Turbo-TMB+sulfuric acid) for peroxidaseactivity conjugated to streptavidin, we observed a significant (100×)signal over background, and the data for triplicate samples ranged from0.006+/−0.0002 to 0.63+/−0.05 absorbance units. In order to determine ifthe TTF1 aptamer “A” would recognize another homeodomain family member,the enzyme-linked assay was used to show that the TTF1 AptamerA does notcross-react with the homeodomain of HOX4 (FIG. 5), nor did the aptamerbind BSA. In addition, an aptamer that was selected for HOX4 binding(not described here) did not cross-react with the TTF1 protein.

Aptamer-enzyme linked assay. To measure the binding of aptamers toproteins immobilized on microtiter plates, 500 ng of purified TTF1 orpurified HOX4 fragment was bound to wells of a Ni-NTA HisSorb plate(Qiagen, Valencia, Calif.) in 200 μL PBS-T for 2 hours at roomtemperature. The wells were then washed 3× with 200 μL PBS-T.Biotinylated aptamers (QIAGEN Operon, Alameda, Calif.) were diluted to 1ng/μL in 200 μL PBS-T, heated to 95° C. and then cooled quickly to 4° C.200 μL of aptamer was incubated with proteins in the HisSorb plateovernight at 4° C. on a plate vortex shaking gently. The wells werewashed 4× with 200 μL PBS-T for 5 min each on a plate vortex.Streptavidin-HRP (Molecular Probes, Eugene, Oreg.) was diluted 1: 10,000into PBS-T and a 200 μL aliquot was incubated with the proteins andbound aptamers in the HisSorb plate for 30 min at room temperature. Thewells were washed again as described above, then 150 μL of Turbo-TMB(Pierce Biotechnology, Rockford, Ill.) was added to each well andincubated for 20 min at room temperature in the dark. The reactions werestopped with the addition of 150 μL of 1 M H₂SO₄ and the protein boundaptamer-streptavidin complex was quantified by determining theabsorbance at 450 nm using a SpectraMax Plus (Molecular Devices,Sunnyvale, Calif.).

Example 3 Determination of the Affinity of Aptamers for TTF1

We utilized surface plasmon resonance employing a BIAcore X instrumentto measure the affinity of the interaction of TTF1 with aptamersimmobilized on a sensor chip. Sensorgrams of a concentration series ofTTF1 injected over TTF1 AptamerA or TTF1 AptamerC are shown in FIGS. 6Aand C, respectively. The affinity, as described by the K_(D), wasdetermined by a global fit using the kinetic simultaneous k_(a)/k_(d)model, assuming Langmuir (1:1) binding. The K_(D) of TTF1 AptamerA forTTF1 was 3.36×10⁻⁹ M, and the K_(D) of TTF1 AptamerC for TTF1 was3.25×10⁻⁸ M. The steady state affinities of TTF1 for the aptamers,determined from plots of Req values derived from sensorgrams in (FIGS.6A and C) fitted locally, correlated well with the simultaneousk_(a)/k_(d) model (FIGS. 6 B and D). K_(D) values for the remainingaptamer sequences (TTF1 Aptamers B, D, E) and the 3 singletons showedthat these aptamers also displayed affinity towards TTF1, ranging from2.2×10⁻⁸ M to 6.7×10⁻⁸ M (not shown).

BIAcore surface plasmon resonance. The affinity of the aptamers fortheir protein targets was measured using surface plasmon resonance (SPR)with a BIAcore X instrument (BIAcore, Piscataway, N.J.). Biotinylatedaptamer (QIAGEN Operon, Alameda, Calif.) was diluted to 0.5 ng/μL inHBS-P (10 mM HEPES, pH 7.5, 150 mM NaCl, 0.05% Tween 20), heated to 95°C., and rapidly cooled at 4° C. before use. Approximately 100 RU ofbiotinylated aptamer (ligand) was immobilized to one flow cell of astreptavidin coated sensor chip. Purified TTF1 protein was diluted intoHBS-P to give a series of concentrations of TTF1 protein (3, 12, 31, 62,125, 250 nM or 2.5, 5, 10, 20, 40, 100 nM) that were injected over thesurface for 2 min at a flow rate of 20 μL/min (to minimize mass transferlimitations). Bulk shift and non-specific interactions with thestreptavidin were subtracted using the response from a reference flowcell. After measuring the off rates for 2 min for each analyteinjection, complete regeneration of the surface was achieved with two 30sec. injections of 0.05% SDS at 50 μL/min. The affinity, as described bythe equilibrium dissociation constant (K_(D)), was determined globallyby fitting to the kinetic simultaneous k_(a)/k_(d) model, assumingLangmuir (1:1) binding. The steady-state affinity was determined fromcurve-fitting to a plot of the Req values, derived from sensorgramsfitted locally, against the concentrations.

Example 4 Comparison of the Specificity of the TTF Aptamer to aMonoclonal Anti-Penta-His Antibody Using Protein Blot Analysis

The results of the enzyme-linked assay suggested that the TTF1 AptamerAexhibited specificity for TTF1. In order to verify the specificity anddetermine whether the aptamer recognized the denatured form of TTF1, aswell as to investigate further the potential uses of the aptamer, weperformed a protein blot analysis (FIG. 7). The TTF1 AptamerA was indeedable to bind the denatured TTF1 on the blot (FIG. 7C lanes 3, 4) andexhibited very little non-specific binding to the proteins in thecleared E. coli lysate or to the purified HOX4 (FIG. 7C lanes 1, 2). Theperformance of the aptamer was similar to the anti-PentaHis antibody interms of chemiluminescent signal intensity and specificity (FIG. 7B).Note that the bands in lane 4 of B and C below the major TTF1 band(marked with an arrow) are degradation products of TTF1 as determined byMALDI mass spectromic analysis (not shown). Also, there is anapproximately 20 kDa protein in the E. coli lysate (FIG. 7C lanes 1, 3)that is recognized by the streptavidin-HRP secondary and not due tocross-reactivity of the aptamer (not shown).

Protein blot analysis with aptamers. Protein samples were prepared forSDS-PAGE by boiling in Laemmli sample buffer and then resolved ondenaturing 4-20% polyacrylamide gels using the mini-Protean 3 system(Bio-Rad, Hercules, Calif.). The proteins were either stained withGelcode Blue (Pierce Biotechnology, Rockford, Ill.) or transferred toPVDF (Schleicher and Schuell, Keene, N.H.). The PVDF membranes wereblocked overnight at 4° C. with 5% BSA in PBS-T, and then probed withbiotinylated TTF1 AptamerA diluted to 1 μg/mL in 5 mL PBS-T for 2 hoursat room temperature with rotation. The blots were washed 3× for 5 minwith 10 mL PBS-T and then probed with Streptavidin-HRP diluted 1:10000in PBS-T. The blots were washed 3× for 5 min before chemiluminescencedetection using pico-west substrate (Pierce Biotechnology, Rockford,Ill.). The blots were imaged using a Fluor-S Multi-Imager (Bio-Rad,Hercules, Calif.).

Example 5 TTF1 Aptamer Affinity Purification

Aptamer affinity chromatography was performed from a complex mixture ofproteins in the soluble fraction of bacterial lysates using biotinylatedaptamers on streptavidin magnetic beads (FIG. 8). Two methods aredescribed, one uses an aptamer that binds a specific protein, TTF1, asan example of purification of untagged proteins from E. coli lysates,and another method that uses an aptamer that binds the His6 tag as anexample of a general use reagent against any His-tagged protein.

Purification of TTF1 native proteins using specific DNA aptamers. TheTTF1 AptamerA was used to purify recombinant TTF1 protein out of the E.coli lysate in a single purification step (FIG. 8). Elution of allproteins bound to the TTF1 AptamerA magnetic beads with SDS, whichremoves all bound proteins from the beads, showed that the purificationof TTF1 was highly specific. Referring now to FIG. 8, a nine laneSDS-PAGE analysis was carried out for aptamer affinity purification ofTTF1 protein from E. coli lysates using biotinylated TTF1 aptamer “A”immobilized on streptavidin magnetic beads. The gel was 4-20%polyacrylamide stained with GELCODE blue. Lane 1 contained clearedlysate from E. coli expressing the protein of interest and lane 2contained the cleared lysate spiked with Ni-NTA purified TTF1 protein(lane 3). Material in lane 4 was from 10 min binding and 2 hr elution at4° C. Material in lane 6 was from 30 min binding and 15 min elution atroom temperature. Material in lane 8 was from 5 min binding and 5 minelution at room temperature. After each elution with Benzonase, anyremaining protein was removed from the aptamer with 0.1% SDS (lanes 5,7, 9).

The generic elution conditions that would be most amenable tohigh-throughput methods were then tested. Elution of the purified TTF1from the affinity matrix was inefficient with 1 M NaCl (not shown);therefore we tested elution with a recombinant DNase. The recovery ofpurified TTF1 with DNase treatment (lanes 4, 6, 8) was approximately25-50% of the total protein bound to the affinity column as revealed bya subsequent denaturing elution with SDS (lanes 5, 7, 9). The efficiencyof elution with DNase was better when the affinity beads were notsaturated with TTF1 protein. This is likely due to the accessibility ofthe aptamer, which may be protected in conditions of saturating amountsof TTF1 protein. Additional optimization for improved elution yield ofspecific proteins without denaturing could be investigated on a case bycase basis. The single-step purification from E. coli furtherillustrates the potential utility of aptamers.

Aptamer affinity purification-TTF1 aptamer. Aptamers immobilized tomagnetic beads were utilized for native protein purification. 10 μg ofbiotinylated TTF1 AptamerA was diluted into 200 μL PBS-T in a PCR tubeand heated to 95° C. for 2 min, then immediately placed at 4° C. for 5min. This material was added to 2 mg M-280 streptavidin magnetic beads(Dynal Biotech, Inc., Brown Deer, Wis.), and 50 μL of 5 M NaCl wasadded, and the mixed for 30 min with rotation at room temperature. Inorder to determine the level of non-specific binding to the M-280 beads,we performed the purification with biotin bound, instead of aptamer. Thebeads were washed 2× with 1 mL PBS-T. 100 μL of cleared lysate from theprotein purification described above was spiked with 10 μg of partiallypurified target protein and then diluted 1:3 with PBS-T. The protein waseluted with DNAse treatment using 12 uL PBS-T containing 50 mM NaCl, 5mM MgCl₂, and 60 units of Benzonase (Novagen, Madison, Wis.). Severalbinding and elution schemes were tested: 1) 10 min binding at 4° C. and2 hour nuclease treatment at 4° C.; 2) 30 min binding at 4° C. and 15min nuclease treatment at room temperature; 3) 5 min binding and 5 minnuclease treatment at room temperature. For each set of conditions, thebeads were washed 4× with 1 mL PBS-T containing 600 mM NaCl, and thenwashed 2× with 1 mL PBS-T containing 50 mM NaCl to adjust the ionicstrength for optimal nuclease activity. Protein that remained afternuclease treatment was removed from the aptamer beads with 12 μL of0.05% SDS. The samples were analyzed by SDS-PAGE on a 4-20% gel that wasstained with GelCode blue (Pierce Biotechnology, Rockford, Ill.).

Example 6 Isolation and Selection of His6 Aptamers and theirCharacterization

The method described below was used to obtain 40 nucleotide DNAoligomers that tightly bind 6 sequential histidine amino acid residues.The target peptide was tagged with biotin to bind the peptide tostreptavidin magnetic beads. These peptide bound magnetic beads werethen used following the method as described herein and in Example 1.While 5 is likely the minimum number of sequential His residues to beused in the affinity tag, more than 6 His residues may readily beemployed. The term “histidine 6” or “His6” or “6His” or “hexahistidine”or “polyhistidine” includes these variations.

Purification of His-tagged proteins using 6His aptamer. The 6H5 aptamerimmobilized to magnetic beads was used to purify 5 different recombinantHis6-tagged proteins out of E. coli lysates with high selectivity in asingle purification step. Elution of all 5 proteins bound to the aptamermagnetic beads was achieved under native conditions using imidazole.

Aptamer Affinity Purification-His6 Aptamer.

The proteins ATF, HNF1b, PBX, TTF1, USF1 were expressed in Rosetta (DE3)pLysS cells (Novagen, Madison, Wis.) induced with 1.0 mM IPTG at 37° C.for 4 hours. One liter of culture grown under these conditions washarvested by centrifugation at 5,000×g for 10 min at 4° C. The pelletwas resuspended in 30 mL of resuspension buffer (50 mM Na₂HPO₄, 300 mMNaCl, 20 mM imidazole, 0.1% Triton X100, 5 mM 2-mercaptoethanol at pH7.0 with 1 mM PMSF Plus (Roche-Applied-Science, Indianapolis, Ind.), and1× Protease Inhibitor Cocktail (Sigma Chemicals, St. Louis, Mo.)). Thecells were then lysed with the Emulsiflex-C5 (Avestin, Ottawa, Ontario,Canada) homogenizer at 15,000 psi. The lysate was centrifuged at15,000×g for 25 min at 4° C. to remove insoluble material.

For each protein sample, 10 μg of biotinylated 6H5 aptamer was dilutedinto 200 μL PBS-T in a PCR tube and heated to 95° C. for 2 min, thenimmediately placed at 4° C. for 5 min. This material was added to 2 mgM-280 streptavidin magnetic beads (Dynal Biotech, Brown Deer, Wis.),plus 50 μL of 5 M NaCl, and mixed for 30 min at room temperature. Thebeads were washed 2× with 1 mL PBS-T before use. 1 mL of E. coli clearedlysate containing the protein of interest was mixed for 20 min at roomtemperature with 2 mg M-280 magnetic beads coated with 10 μg of 6H5aptamer or with 35 μL of Ni-NTA magnetic beads (Qiagen, Valencia,Calif.). The beads were washed 3× with PBS-T containing 500 mM NaCl.Proteins were eluted with 20 uL PBS-T containing 1 M imidazole.Replicate samples were treated with 0.05% SDS to elute all proteins fromthe beads in order to determine the effectiveness of the imidazoleelution. 10 μL of this material was analyzed by SDS-PAGE on a 4-20% gelthat was stained with GELCODE blue.

The degenerate sequences of the His6 aptamers isolated are disclosedbelow:

Aptamer 6H5 (approx. 30% of total) SEQ ID: NO: 14GGCTTCAGGTTGGTCTGGTTGGGTTTGGCTCCTGTGTACG Aptamer 6H1 (approx. 20% oftotal) SEQ ID: NO: 15 GGCAAAAAGGATTGCCCAGGTCTGCTGTCTAGCCGGATTC Aptamer6H7 (approx. 8% of total) SEQ ID: NO: 16GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC

The sequences, SEQ ID NOS: 14, 15 and 16, were flanked on either ends bythe LIC primers (SEQ ID NO: 17 and SEQ ID NO: 18). The percentage oftotal indicates the percentage of the total final aptamer mixturerepresented by the particular aptamer, after the complete iterativeseries of binding, elution and amplification.

FIG. 9 shows a multiple sequence alignment using AlignX (Vector NTI), amodified Clustal W algorithm, of the 3 aptamers that were selected forbinding to hexahistidine. The 40 bases of unique sequence, and not theflanking LIC primer sequences, are in the alignment. The baseshighlighted in black are found in all three aptamer sequences, and thebases highlighted in grey are found in two of the three aptamersequences. A consensus pattern generated from the multiple sequencealignment is shown, and is GGCTANNNGGGTTGGTCTGGTTGGGTTTGGCNCCGGNNTCNG(SEQ ID NO: 21). The aptamer sequences are 31.8% identical and 79.5%conserved.

The selected His6 aptamer sequences were characterized to determine thebinding affinity of the aptamer sequence to various target molecules,the kinetics of the good binding affinity and the optimized method ofaffinity purification. Table 1 summarizes the characterization ofselected His6 sequences, 6H1, 6H5 and 6H7. The aptamers were furthercharacterized according to the aptamer binding affinity to known andcommon proteins using surface plasmon resonance.

The kinetics of the interaction were determined using the methoddescribed in Example 3, i.e. surface plasmon resonance (SPR). Thebiotinylated aptamer to a His6 peptide was immobilized to one flow cellof a streptavidin coated sensor chip. Purified His6 tagged protein wasdiluted to give a series of concentrations that are injected over thesurface. After measuring the on and off rates, the affinity, asdescribed by the equilibrium dissociation constant (K_(D)), isdetermined globally by fitting to the kinetic simultaneous k_(a)/k_(d)model. This is preferably measured by SPR in a device such as a BIAcoreor by equilibrium dialysis. The following table describes thecharacterization of the His6 aptamers.

TABLE 1 Summary of His6 aptamer characterization Affinity AptamerProtein Binding (SPR) Kinetics (SPR) Purification 6H5 PBX high, unusualno fit, multimer ATF, HNF1b, USF1 good, dimer K_(D) = 176 nM PBX, TTF1,XF1293 good K_(D) = 30 nM USF1 IDH poor 6H1 USF1 good, dimer K_(D) = 36nM XF1293 very good K_(D) = 0.08 nM 6H7 XF1293 good K_(D) = 0.4 nM PBXhigh, unusual no fit, multimer XF0749 good K_(D) = 42 nM

In the above Table, the first column is the designation of the aptamer,as represented in the above sequences. The next column, “Protein”represents the protein which was his-tagged and tested for binding tothe aptamer. The proteins may be further identified as follows:

ATF—NP 005162, activating transcription factor 1 (Homo sapiens)

HNF1b—NP 033356, hepatocyte nuclear factor-1 beta (Homo sapiens)

PBX—P41778, Pre-B-cell leukemia transcription factor-1 (cionaintestinalis)

USF1—P22415, upstream stimulatory factor (ciona intestinalis)

TTF1—CAA08756, thyroid transcription factor (ciona intestinalis)

XF1293—NP 298582, hypothetical conserved (Xylella fastidiosa)

IDH—E. coli isocitrate dehydrogenase

XF0749—NP 298039, virulence regulator (Xylella fastidiosa)

In the third column, “Binding,” the binding of the aptamer to thespecific protein, as measured by SPR is characterized. In the case ofPBX, for example, the binding was “unusual” in that the data curvescould not be fitted because they were 3-4 times the expected response,possibly due to multimerization, aggregation, precipitation or otherevents that would affect the binding response. The fourth column,“Kinetics,” represents the binding of the aptamer to the protein, asmeasured by SPR. The K_(D) is a measure of the equilibrium dissociationconstant, which describes the “on and off” rates” of the protein-aptamercomplex.

Finally, the fifth column, “Affinity purification” shows that aptamer6H5 was successfully used to affinity purify the listed, tagged proteinsby binding to their His6 tag. Each his-tagged protein was purified froman E. coli lysate.

Example 7 Use of His6 Aptamers as Capture Agents for SPR or MicroarrayFormat

DNA aptamers are ideal reagents for SPR due to their stability andoptimal binding characteristics. We have shown that our 6H5 aptamerworks well as a capture agent. FIG. 10 shows schematics for capturemethods using the His6 aptamers of Example 6 for SPR experiments. FIG.10A shows the traditional method using an antibody that binds to theHis6 tag. FIG. 10B shows the use of a biotinylated aptamer on astreptavidin chip that binds to the His6 tag. FIG. 10C shows the use ofa biotinylated linker primer on a streptavidin chip that binds to thefixed portion of the aptamer by base complementarity.

Preliminary data (not shown) has shown that most commercially availableantibodies (such as PENTAHIS antibody from Qiagen, Valencia, Calif.)display fast off rates that causes baseline drift as the capture proteindissociates from the antibody. It was found that an alternative antibodyfor the 6His tag (from Covance Research Products, Denver, Pa.) that doesnot result in significant baseline drift, as described above. TheCovance antibody (amine coupled to a BIAcore CM-5 chip) was compared tothat of our 6H5 aptamer (SEQ ID NO: 14) (bound to a streptavidin-coatedBIAcore chip due to an attached biotin moiety) (FIG. 11).Molar-equivalent amounts of antibody and aptamer bound to the sensorchip (80 fmoles and 72 fmoles, respectively). Samples containing 3concentrations of His-tagged protein were passed over the chips, andbinding and dissociation characteristics were monitored. While theantibody-coated chip bound more protein, comparison of the dissociationrates suggest that the aptamer displays lower baseline drift (FIGS. 12and 13). This, coupled with the greater stability of the aptamer,demonstrates that the 6H5 aptamer is an ideal capture agent for SPR.

Example 8 Novel Capture Scheme for Aptamer (for SPR or Microarray ChipSurface)

A disadvantage to both strategies, described above, is that the captureagent is irreversibly coupled to the SPR chip (BIAcore, Piscataway,N.J.), making the chip unable to be reused with another capture agent.When using an aptamer or antibody that binds a generic tag, such as the6His tag, this is less of a disadvantage since the chip can be used withadditional proteins that contain the tag. However, if the experimentinvolves testing a number of antibodies or aptamers, the chip becomessingle use. Additionally, there is an added cost associated withgenerating biotinylated aptamers (as opposed to non-biotinylated ones),therefore it would be advantageous to have a method to couplenon-biotinylated aptamers to surfaces such as SPR chips. In a microarrayformat, this is a major advantage since a large number of aptamers wouldbe bound to the slide surface, which could be made by PCR or oligosynthesis without extra cost.

Herein is described a method to attach non-biotinylated aptamers tocoated surfaces, specifically streptavidin-coated surfaces. Abiotinylated “linker primer” oligonucleotide was obtained, whichcontained sequences complementary to portions of the fixed 3′ primersequence of the His6 aptamer sequences. As shown in FIG. 10C, the linkerprimer binds to a streptavidin-coated sensor chip via the biotin moiety,and captures the aptamer via its complementary sequences.

Data shows that the linker primer binds to the BIAcore streptavidin chipat a capacity (saturation) equal to that of the DNA aptamer (52 fmoles,1.3 ng). When the linker is bound at saturating concentrations,preliminary data suggested that approximately 30% of the linker isaccessible for aptamer binding. Dissociation of the aptamer from thelinker, however, is extremely slow, displaying characteristics similarto that of the biotinylated aptamer bound directly to the streptavidinchip (2.5 RU/min) (FIG. 14).

Many SPR applications (such as the determination of binding constants)require that sub-saturating concentrations of capture molecule be used.The linker primer bound at sub-saturating concentrations (0.06 ng) tothe sensor chip can bind aptamers at a ratio of approximately 1:1,indicating that the nearly all the linker is accessible as a captureagent (FIG. 15). The aptamer can be removed from the linker with 50 mMNaOH, 1M NaCl, leaving a linker that is able to rebind aptamerefficiently. The bound aptamer is able to capture its protein partner,which can subsequently be removed from the aptamer/linker pair byaddition of 0.05% SDS, leaving an aptamer that retains the ability tobind its protein partner.

While the present compositions and processes have been described withreference to specific details of certain exemplary embodiments thereof,it is not intended that such details be regarded as limitations upon thescope of the invention. The present examples, methods, procedures,specific compounds and molecules are meant to exemplify and illustratethe invention and should in no way be seen as limiting the scope of theinvention. Any patents or publications mentioned in this specificationand below are indicative of levels of those skilled in the art to whichthe invention pertains and are hereby incorporated by reference to thesame extent as if each was specifically and individually incorporated byreference.

1. A method for obtaining an aptamer sequence having high affinity to atarget molecule, comprising: (a) providing a target molecule with apolyhistidine tag, said polyhistidine tag having a binding affinity tomagnetic beads; (b) binding the target molecule to magnetic beads via a6His aptamer, wherein the magnetic beads are coated with the 6Hisaptamer comprising the aptamer sequence of SEQ ID NOS: 14, 15, 16 and/or21; (c) providing a library of degenerate aptamer sequences, whereineach of said degenerate aptamer sequences is flanked by fixed sequenceswhich permit ligation-dependent cloning; (d) contacting the magneticbead-bound target molecule with said library of degenerate aptamersequences to allow binding of said degenerate aptamer sequences to thetarget molecule with high affinity, wherein said high affinity has adissociation constant of at least 10⁻⁴M; (e) forming magnetic bead-boundtarget-aptamer sequence complexes upon binding of said degenerateaptamer sequences to the target molecule with high affinity; (f)separating said magnetic bead-bound target-aptamer sequence complexesfrom the library of non-binding degenerate aptamer sequences; (g)separating target-bound degenerate aptamer sequences from said magneticbead-bound target aptamer sequence complexes to form a pool of highaffinity binding aptamer sequences; (h) amplifying the high affinitybinding aptamer sequences; and (i) iteratively repeating steps (b)through (h) a sufficient number of times to result in identification ofat least one high affinity binding aptamer sequence having high affinityfor the target molecule, thus obtaining an aptamer sequence having highaffinity to said target molecule.
 2. The method of claim 1 furthercomprising: (j) subcloning the high affinity binding aptamer sequenceinto a plasmid and transforming E. coli; (k) isolating said plasmidcontaining said high affinity binding aptamer sequence from transformedE. coli; and (l) amplifying and sequencing said cloned high affinitybinding aptamer sequence.
 3. The method of claim 1 wherein said flankingfixed sequences are primer sequences.
 4. The method of claim 2 whereinsaid amplification is by rolling circle amplification.
 5. The method ofclaim 1, wherein the 6His aptamer consists of SEQ ID NO: 21 and whereinSEQ ID NO: 21 exhibits binding activity to polyhistidine tags.
 6. Themethod of claim 1, wherein the 6His aptamer consists of SEQ ID NO: 14and wherein SEQ ID NO: 14 exhibits binding activity to polyhistidinetags.
 7. The method of claim 1, wherein the 6His aptamer consists of SEQID NO: 15 and wherein SEQ ID NO: 15 exhibits binding activity topolyhistidine tags.
 8. The method of claim 1, wherein the 6His aptamerconsists of SEQ ID NO: 16 and wherein SEQ ID NO: 16 exhibits bindingactivity to polyhistidine tags.